Marek's disease is a malignant, lymphomatous disorder of chickens caused by a herpesvirus, Marek's disease virus. The virus infects chickens and results in the development of T-cell lymphomas in a variety of tissues in the weeks following infection, which ultimately results in the death or condemnation of the infected chickens at processing. The disease is unique among herpesvirus-induced disorders in that it has been controlled by the poultry industry for twenty years by vaccination of all commercial broilers and broiler breeders in the United States and many parts of the world.
MDV is a DNA virus having an envelope and is classified in the family Herpesviridae. It is further classified into the following three serotypes:
Type I: virulent strains of MDV which are pathogenic and tumorigenic to chickens, and attenuated nonpathogenic strains derived therefrom. PA1 Type II: naturally occurring nonpathogenic strains of MDV; and PA1 Type III: herpesvirus of turkeys ("HVT") strains, which are nonpathogenic to chickens.
The pathogenesis of MDV infection and the classical virology of MDV have been studied throughout the twentieth century. Progress on the molecular analysis of MDV has been made during the last decade. Important advances include cloning of the viral DNA molecule (Fukuchi et al., J. Virol. 51:102-109, 1984), the generation of monoclonal antibodies against MDV; the identification of viral polypeptides; the generation of transcription maps (Schat et al, Int. J. Cancer 44:101-109, 1989), and the identification of genes on the MDV genome (Buckmaster et al, J. Gen. Virol. 69:2033-2042, 1988). To date there has been virtually no genetic analysis of the virus, although one phosphonoacetate-resistant mutant of HVT has been reported.
Marek's disease is of tremendous economic importance and effective Marek's disease vaccines are critical to the livelihood of the poultry industry. The most widely used vaccine is HVT, although currently in many regions chickens are vaccinated with a combination of MDV vaccine strains. The existing Marek's disease vaccines are unlikely to remain adequate in the future and the development of recombinant Marek's disease vaccines continues to be an important challenge to researchers in the field. Because existing Marek's disease vaccines have already been used for twenty years in the poultry industry as live herpesvirus vaccines, they are currently being researched as potential herpesvirus vectors suitable for poultry.
Virus vectors have been reported using, for example, vaccinia, papillomavirus, baculovirus, parvovirus and tobacco mosaic virus. All have been reported as a cloning vector or an expression vector for a foreign gene. MDV has also been reported as an expression vector for a foreign gene, as described by T. Ishikawa et al. in European Patent Application 0 334 530. This application describes inserting a foreign gene, such as the gene coding for hemagglutinin and neuraminidase of Newcastle disease virus ("NDV") into the gene encoding the A antigen site (gp 57-65 gene) of HVT. The recombinant HVT is used to produce a vaccine to both NDV and MDV.
In WO 88/07088, S. Martin et al. describe the method of inserting a foreign gene into a nonessential region of HVT and infecting the bird with the viral vector which will ultimately produce an immunogenic reaction to both the foreign gene product and the HVT. In particular, HVT is the only avian herpesvirus taught as a viral vector, and the nonessential regions used encode for thymidine kinase and for 1-.beta.-D-arabinofuranosylthymine resistance.
Cochran et al., in PCT application WO 89/0140, describe insertion of foreign DNA into attenuated herpesvirus vectors. They describe a recombinant fusion protein comprising an antigenic amino acid sequence fused to a portion of the gpX glycoprotein from pseudorabies virus. A cDNA copy of the large segment of RNA of infectious bursal disease virus ("IBDV"), which encodes three polypeptides, namely VP2, VP4 and VP3, and the E. coli .beta.-galactosidase gene were inserted into a nonessential site within the unique long region of the HVT genome. This recombinant virus was used as a vaccine to IBDV. MDV A antigen gene (gp 57-65) was inserted into the same site of HVT in order to produce an improved vaccine against MDV.
Although genetic analysis of some herpesvirus, including herpes simplex virus and pseudorabies, has been done, the genetic structure of the DNA genome of MDV serotype 1 ("MDV-1") is not well known.
Current vaccines against the various poultry diseases are often produced through the use of live, attenuated pathogens, which pose a risk of inoculating animals with inadequately attenuated pathogenic microorganisms.
Inactivated vaccines generally induce only a low level of immunity requiring additional immunizations. Furthermore, the neutralization-inducing antigenic determinants of the viruses may become altered by the inactivation treatment, decreasing the protective potency of the vaccine.
When more than one attenuated, live pathogen is combined in a vaccine, another problem may arise. The mutual influence of the antigenic components may result in a decrease in the immunogenicity of one or more of the constituent antigens.
In order to produce a potent vaccine to Marek's disease and at least one other avian disease, through the use of an MDV vector wherein the DNA genome of the MDV contains a foreign gene that encodes an antigen from another avian disease causing agent, a nonessential region of the MDV genome must be found and used as an insertion region. Once the foreign gene is inserted into the insertion region, the corresponding gene product must be expressed. The MDV vector will, once given to chickens, elicit an immune response to both MDV and the foreign gene product, such as a protein, preferably with a greater potency than that exhibited by a combined vaccine.